Regulation of Megakaryocytopoiesis and Platelet Production by Tyrosine Kinases and Tyrosine Phosphatases

METHODS: A Companion to Methods in Enzymology 17, 250 –264 (1999) Article ID meth.1998.0735, available online at http://www.idealibrary.com on

Regulation of Megakaryocytopoiesis and Platelet Production by Tyrosine Kinases and Tyrosine Phosphatases Hava Avraham1 and Daniel J. Price Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, Massachusetts 02115

Megakaryocytopoiesis is the process by which bone marrow progenitor cells develop into mature megakaryocytes, which in turn produce platelets required for normal hemostasis. The development of this hematopoietic lineage depends on a variety of growth factors and cytokines. Growth factor-dependent tyrosine kinase receptors important in megakaryocytopoiesis include c-Kit, fibroblast growth factor receptor, the RON receptor, and the macrophage colony-stimulating factor receptor. Binding of growth factors to their respective receptors results in receptor dimerization and subsequent autophosphorylation on tyrosine residues. Tyrosine autophosphorylations become sites of association for cytoplasmic signaling molecules via their SH2 domains. Some of these molecules are themselves cytoplasmic tyrosine kinases such as the Src kinases, TEC, and CHK. Others are molecules such as phospholipase C-g, phosphoinositol 3-kinase, Shc, GTPase-activating protein, and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. These molecules generate second messengers, regulate the phosphorylation of other downstream molecules, and also regulate the phosphorylation of the receptor itself. The different cytoplasmic components activate pathways involved in either changes in cell growth or changes in the cytoskeleton that affect maturation of the cell. Cytokine receptors also generate signals involved in growth and differentiation. Some of these second messengers overlap with those of the receptor tyrosine kinases. Others, such as the JAKs/STATs, are involved in transcriptional control and are unique to the signaling mediated by cytokine receptors. We describe the contribution of these different signals to the growth/differentiation

1 To whom correspondence should be addressed at Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115. Fax: (617) 975-5240. E-mail: havraham@caregroup. harvard.edu.

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processes of megakaryocytes. We also describe the contribution of receptor and nonreceptor tyrosine phosphatases to these processes. Lastly, we have compiled selected methods related to the study of protein phosphorylation in megakaryocytes. © 1999 Academic Press

The process of bone marrow megakaryocytopoiesis leading to platelet formation involves a lineage-specific development of bone marrow stem cells through a series of cell types, finally resulting in mature megakaryocytes, which then fragment into functional platelets. As shown in Fig. 1, this process is highly dependent on the cellular action of specific cytokines and growth factors. Some of these factors are soluble, and some are presented on the surface of other bone marrow cells. The result of growth factor and cytokine action is a specific change in the pattern of tyrosine phosphorylation within the cells, leading to other downstream effects that eventually lead to proliferative and/or differentiative effects. As shown in Fig. 2, the activation of receptor tyrosine kinases results in mobilization and subsequent activation of signaling molecules such as phospholipase C-g (PLC-g), Raf-1 kinase, MAP kinases, phosphatidylinositol 3-kinase (PI3-kinase), GTPase-activating protein (GAP), p21ras, and Src family kinases. On activation, cytokine receptors such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3), and thrombopoietin (TPO) become phosphorylated by cytoplasmic tyrosine kinases, the Janus kinases (JAKs), leading to the association of their substrates, signal transducers associated with transcription (STATs) (not shown). 1046-2023/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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In this review, we will summarize the current body of knowledge relating to growth factor- and cytokinemediated tyrosine phosphorylation in cellular processes related to megakaryocytopoiesis. Although most of the effects involve regulation of tyrosine kinases, we will also include the effects of tyrosine phosphatases on these cellular processes. We will explain how a diverse and coordinated array of growth factors and cytokines leads to specific tyrosine phosphorylation, resulting in cytoplasmic signaling including serine/threonine phosphorylation and other related second messengers. We will then show how these signals result in specific transcriptional effects leading to megakaryocytic proliferation and differentiation. Also included in this review will be a selected compilation of methods used in work relating to tyrosine phosphorylation and megakaryocytic growth and differentiation. These methods will include specific applications of expression systems for overexpression of mutant and wild-type genes, determination of sites of SH2 binding to receptors, and determination of in vivo sites of phosphorylation. Finally, we will describe the use of transgenic mice for investigation of megakaryocytic signaling components related to tyrosine phosphorylation.

ROLE OF RECEPTOR TYROSINE KINASES IN MEGAKARYOCYTE GROWTH AND DIFFERENTIATION Much of the stimulation of megakaryocytic growth and differentiation is influenced by specific growth

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factors that act on the megakaryocytic lineage as shown in Fig. 1. On ligand binding, these receptors dimerize and undergo tyrosine autophosphorylation (Fig. 2). The effects of specific receptor tyrosine kinases on megakaryocytic development and the molecular mechanisms involved are detailed in the following sections. c-Kit Receptor Tyrosine Kinase and Stem Cell Factor/ Kit Ligand (SCF/KL) The c-Kit proto-oncogene is a transmembrane receptor tyrosine kinase of 145 kDa with sequence homology closely related to the platelet-derived growth factor receptor and macrophage-colony stimulating factor-1 (1– 4). In mice, dominant-negative mutations of either c-Kit (W) or its ligand SCF/KL (Sl) give rise to defects in hematopoiesis, gametogenesis, and melanogenesis (5– 8). Administration of SCF/KL to mutant Sl/Sl mice was able to reverse and actually enhance the platelet production in these mice (9). SCF/KL was also able to increase proliferation of megakaryocytic progenitors in a synergistic manner in combination with GM-CSF, IL-3, IL-6 (10), and TPO (11). Studies of cord blood hematopoietic colony formation in methylcellulose cultures in combination with IL-3, IL-6, and IL-11 have shown that SCF/KL induced proliferation of primitive megakaryocytic progenitors, but did not promote megakaryocytic maturation (12). Thus, SCF/KL did not increase the ploidy of megakaryocytes, but did have an effect on proliferation. SCF/KL appeared to have its greatest effect on undifferentiated myeloid progenitors and BFU-MK

FIG. 1. Growth factors involved in megakaryocyte growth and maturation. Cell types are as follows: HPP-CFC, high proliferative potential colony-forming cell; HPP-CFU-MK, high proliferative potential colony-forming unit-megakaryocyte; BFU-MK, burst-forming unit-megakaryocyte; CFU-MK, colony-forming unit-megakaryocyte.

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cells, while CFU/MK and mature megakaryocytes were less responsive. SCF/KL appears to have a pleiotropic function in megakaryocyte development. This is in part due to its presence in soluble and membrane-bound forms (13, 14). It has been shown that SCF/KL in its membrane-bound form, on bone marrow stromal fibroblasts, mediated the association of these fibroblasts with megakaryocytes and resulted in an increased DNA synthesis in the megakaryocytes. It has also been shown that SCF/KL action could result in increased platelet aggregation by binding to and activating c-Kit that was present in platelets (15). Thus, SCF/KL in binding to c-Kit can serve both growth factor and cellular adhesive functions that contribute to increased hematopoiesis and hemostasis. The molecular basis of c-Kit signaling is in general characteristic of receptor tyrosine kinases of the PDGF receptor family. Molecules that are known to interact with this class of receptor include PI3kinase (16), PLC-g1 (17), Src family kinases (18, 19), GTPase-activating protein (20), and the tyrosine phosphatase SHP-1 (21–23). Other associations include the tyrosine kinase TEC (24), Shc (19) and the Csk-related kinase CHK (19). Most of these associations, with the exception of TEC, which is constitutively bound, are mediated by transient tyrosine autophosphorylations of c-Kit interacting directly with SH2 domains on the respective signaling molecules. It has also been recently documented by members of our laboratory that the receptor tyrosine phosphatase PTP-RO is constitutively associated with c-Kit and becomes tyrosine-phosphorylated

upon SCF/KL stimulation (25). In many cases, the sites of association of these signaling molecules have been determined by peptide competition, peptide binding, or site-directed mutagenesis. The downstream effects of some of these signaling molecules will be detailed in the section on cytoplasmic tyrosine kinases and tyrosine phosphatases. As mentioned above, the functions of c-Kit relate to both cellular growth and cell–matrix adhesion. It has been shown recently by Kinashi et al. (26) that the association of PI3-kinase and PLC-g with activated c-Kit leads to cellular adhesion. Using the PDGF receptor transfected into mast cells, it was shown that this receptor, like c-Kit, was able to mediate both mast cell adhesion to fibronectin and cellular growth. A double mutant, unable to associate with PI3-kinase and PLC-g, was no longer able to mediate cell adhesion, but was able to mediate cell growth. This is in agreement with the finding of Courtneidge et al. (27), who found that in the case of the m-CSF receptor, PI3-kinase did not mediate growth, while Src kinase activated by this receptor did affect this function. In addition to the tyrosine autophosphorylation of c-Kit, which has been documented at specific sites, there is specific serine phosphorylation that is mediated by protein kinase C and acts in an SCF/KL stimulated negative feedback loop to down-regulate c-Kit (28, 29). These serine phosphorylations account for 60% of the total SCF/KL stimulated phosphorylations and account for up to 90% of the phosphorylations in the resting cell. By peptide mapping, it has been shown that phosphorylation of c-Kit by

FIG. 2. Binding of growth factors to receptor tyrosine kinases leading to receptor associations and downstream signaling.

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protein kinase C at Ser-741 and Ser-746 is an important mechanism for down-regulating the tyrosine autokinase activity and the association of signaling molecules (30). Fibroblast Growth Factor Receptor and Its Ligand FGF FGF receptors have been characterized as being of four different isotypes, FGFR-1 (flg) (31), FGFR-2 (bek) (32), FGFR-3 (33), and FGFR-4 (34). They are transmembrane receptor tyrosine kinases having molecular masses of ;150 kDa and are known to contain heparin sulfate residues on their extracellular domains. Of the fibroblast growth factors, basic fibroblast growth factor, a protein of 154 amino acids, appears to be the major product secreted from fibroblast cells of bone marrow stroma and a variety of other tissues (35–38). Basic fibroblast growth factor, like SCF/KL, is able to stimulate growth of early marrow progenitors in combination with other cytokines such as erythropoietin and G-CSF (35–37, 39 – 42). Megakaryocytes have been shown to express FGFR-1 (flg) and FGFR-2 (bek) (42). It was recently shown by members of our laboratory that bFGF causes the increased proliferation of a variety of megakaryocytic cell lines (43) and also causes an increased CFU-MK colony size (cells/colony) in primary megakaryocyte cultures, in combination with GM-CSF and IL-3. Basic fibroblast growth factor also increased the adhesion of megakaryocytic cell lines CMK and MEG-01 as well as primary megakaryocytes to bone marrow fibroblasts. Treatment of primary human megakaryocytes with bFGF also resulted in the increased secretion of IL-6, TNFa, and GM-CSF by these cells. As with other receptor tyrosine kinases, bFGF binding led to autophosphorylation and association of signaling molecules via SH2 domains. A major FGF receptorassociated protein is phospholipase C-g, which has been shown to associate specifically with Tyr-766 of the activated FGFR by peptide competition and sitedirected mutagenesis (44, 45). Interestingly, when the Y766F FGFR mutant was transfected into Ba/F3 hematopoietic cells, there was an approximate threefold reduction in the activation of Raf-1 and MAP kinase. Phosphopeptide maps of Raf-1 indicate dual regulation by both ras and PLC-g, leading to Raf-1 activation and increased proliferation (46). Other signaling molecules such as PI3-kinase and GTPase-activating protein appear not to be associated with the activated FGF receptor (45). The tyrosine phosphatase SHP-2 has been shown to be

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associated with the FGF receptor in a number of systems (47, 48). The injection of a dominantnegative SHP-2 mRNA into Xenopus oocytes blocked FGF-mediated induction of mesoderm and FGFinduced MAP kinase (47). This outcome was in agreement with studies of SHP-2 dominantnegative transgenic mice in which failure to activate MAP kinase resulted in death in utero due to defects in notochord formation and posterior elongation (48). Thus, signaling even of one element of a receptor-mediated pathway can often be dependent on a variety of associated regulatory factors. The RON Receptor and Its Ligand, MacrophageStimulating Protein (MSP) The RON receptor is a heterodimeric transmembrane receptor consisting of a 285 amino-acid extracellular a subunit that is disulfide linked to a 1069 amino-acid transmembrane b subunit. This protein is closely related to the receptor tyrosine kinase c-MET (p190met), which binds to hepatocyte growth factor (HGF) (49, 50). The ligand for the RON receptor is MSP, an 80-kDa serum protein that was originally found to be a chemotactic factor for macrophages in mice (51–53). Stimulation of primary megakaryocytes as well as DAMI and CMK cell lines resulted in maturation as seen by an increase in ploidy (54). Significantly, the action of MSP was shown to be mediated by the production of IL-6 by megakaryocytes. The activation by MSP was specifically toward the RON receptor, and there was no activation of the related c-MET by MSP. Unlike SCF/KL and bFGF, there was no synergism of RONmediated maturation by GM-CSF, IL-3, or IL-6. Although very little is known about the signaling from RON leading to IL-6 secretion, it is known that PI3-kinase becomes activated and associated with RON following autophosphorylation of the receptor (55). Inhibition of PI3-kinase signaling by wortmannin abolished the MSP-induced migration of epithelial cells, indicating that at least this function requires signaling through PI3-kinase. In Ba/F3 cells, RON overexpression resulted in MSP-dependent cell growth while in an erythroleukemia cell (MEL/ STK), MSP stimulated apoptosis (56). Apoptosis was accompanied by a prolonged stimulation of c-Jun N-terminal kinase (JNK). In these systems, signaling molecules associated with activated RON were PLC-g, PI3-kinase, Shc, and Grb2. Mutation of Y1330 and Y1337 was sufficient to abolish both growth and apoptotic effects in these systems. MSP has also

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been reported to inhibit proliferation of megakaryocytic Mo7e cells (57). These investigators also showed that MSP antagonized the colony-forming synergistic effects of SCF/KL or FLT-3 plus GMCSF, G-CSF, or IL-3 on bone marrow myeloid progenitors. Taken together with the data on CMK and DAMI cells (54), it appears that stimulation of RON by MSP results in a growth inhibitory/maturation effect, contrasting with c-Kit, which produces a growth stimulatory or self-renewal function. Macrophage Colony-Stimulating Factor (m-CSF) and m-CSF Receptor The m-CSF receptor is present primarily on monocyte–macrophage hematopoietic lineages, placental trophoblast cells, and osteoblasts (58). While the m-CSF receptor is not present on mature megakaryocytes (59), treatment of thrombocytopenic cancer patients with m-CSF has been shown to enhance the production of monocytes, neutrophils, and platelets in these patients (60). Thus, it was suggested that m-CSF indirectly increased platelet production by stimulating the production of megakaryocytopoietic cytokines such as GM-CSF, G-CSF, and megakaryocyte potentiator by the mature monocytes. This receptor tyrosine kinase of molecular mass 160 kDa, the cellular homologue of the retroviral oncogene, v-fms, is most closely related to c-Kit and the PDGF receptor and interacts with many of the same signaling components (27, 61– 63). Experiments in 5-fluorouracil-treated myelosuppressed mice have shown that treatment with m-CSF in combination with IL-1 stimulated early events in platelet repopulation. Other cytokines such as IL-6, IL-3, and GM-CSF were required as secondary cytokines in platelet repopulation (64).

CYTOPLASMIC TYROSINE KINASES INVOLVED IN MEGAKARYOCYTE GROWTH AND DIFFERENTIATION Src Family Kinases Src family kinases have been implicated in a variety of systems where cellular growth is regulated. Frequently, the Src kinases become activated by association with receptor tyrosine kinases (19, 27, 65– 68). Src kinase stimulation via the PDGF and m-CSF receptors has been implicated in the stimulation of DNA synthesis as was mentioned in the

previous section. Stimulation of DNA synthesis has been shown to involve signaling through c-Myc but not Fos/Jun pathways in PDGF-stimulated NIH 3T3 fibroblasts (69). The situation in megakaryocytic proliferation may be slightly more complex. In studies done in our laboratory, we have found that .80% of the Src family kinase activity in Mo7e cells is p56/p53 Lyn, with the remaining activity being Hck and Fyn. When we examined Lyn activity in response to SCF/KL, we observed an ;50% stimulation of the total Lyn kinase activity (submitted for publication). Since there was a high basal activity, it was possible that sites other than the c-Kit receptor were involved in Lyn regulation. It has been shown by Li et al. (70) that Lyn is constitutively associated with the b subunit of the GM-CSF receptor in Mo7e cells. On treatment of serum-starved Mo7e cells with GM-CSF, Lyn activity increased by approximately fivefold. These same cells showed changes in DNA synthesis and proliferation that were consistent with the stimulation of Lyn activity. Thus, it is possible that, in megakaryocytes, parallel pathways exist for Lyn or other Src kinase stimulation. It may be that while some cells respond more to growth factors such as SCF/KL and bFGF, others respond to cytokines such as GM-CSF. The distinction between these two receptor pathways appears to be that different signals accompany the stimulation of Lyn activity. Receptor tyrosine kinases stimulate Src kinases important for DNA synthesis (65– 68) while PI3-kinase and PLC-g appear to mediate cell adhesion functions (26). Cytokine receptors such as GMCSF stimulate Src kinases plus factors such as JAK/ STATs, which are involved in selective transcriptional activity. For a review of signaling through JAK/STATs, see Ihle (71). Thus, particular growth factors can stimulate cellular growth by increasing the level of Src kinase activity, and this proliferation is accompanied by other traits that are determined by coexpressed receptor activities. Csk Homologous Kinase The Csk homologous kinase (CHK) is an SH2/ SH3-containing tyrosine kinase recently identified by our laboratory and by others (72–79). It has a molecular mass of 56 kDa and bears ;50% identity to the Csk tyrosine kinase. Like Csk, CHK phosphorylates the C-terminal tyrosine of Src family kinases, resulting in the inactivation of these enzymes. CHK is expressed almost exclusively in megakaryocytes and in brain. It has also been re-

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ported that CHK is expressed in activated T cells and in monocytes (79). It was found that while levels of Csk remained relatively constant, the levels of CHK protein and mRNA increased in response to SCF/KL in megakaryocytic Mo7e cells (80). We have also found that CHK is present in a variety of tumor tissues at levels significantly higher than in the normal adjacent tissue. These tumors include breast cancer (81), ovarian cancer, and prostate cancer (D. Price and H. Avraham, unpublished results, 1997). Studies of Davidson et al. (82) involving selective expression of CHK or Csk in a Csk-deficient fibroblast indicated that both enzymes phosphorylated and inactivated Src kinase in situ. The only differences that were seen involved the inability of CHK to substitute for Csk in certain T cell-receptormediated functions in the T cell line, BI-141. Studies in our laboratory suggest that CHK binds to a number of different growth factor receptors including c-Kit (19, 83), PDGF receptor (D. Price unpublished results), ErbB-2 (81, 84), and TrkA (85). In all of these cases, the association has been shown to be via SH2 association with a phosphotyrosyl residue on the activated receptor. CHK was found to associate with the diphosphorylated sequence 568Y*VY*IDPT of c-Kit (19). This is the same site with which the Src kinases Fyn and Lyn have been shown to associate (19, 86). Our experiments indicated that Csk was unable to associate with this site. We have hypothesized that the coassociation of CHK, Lyn, and c-Kit may be the means by which CHK regulates Lyn in megakaryocytes. We have demonstrated by overexpressing CHK in Mo7e cells via a recombinant vaccinia virus that SCF/KL-stimulated Lyn activity was suppressed (86). Thus, it appears that one function of CHK in megakaryocytes is the modulation of Lyn kinase activity. It is possible that CHK performs functions related to other growth factor receptor-associated components. CHK may also regulate Src kinases in other subcellular locations. As yet, there is no indication that CHK could phosphorylate other non-Src family kinase substrates, but this possibility cannot be ruled out. It has also been shown that CHK functions in the mature platelet to regulate Lyn function during thrombin activation (87). In nonactivated platelets, CHK was associated with Lyn in the Triton X-100 soluble fraction where CHK is closely associated with CD36. After 10 s of platelet activation by thrombin, CHK was translocated to the Triton X-100 insoluble fraction and away from Lyn. This translocation resulted in activation of Lyn, presumably because CHK was not in

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close proximity to it and could not carry out C-terminal phosphorylation. In these experiments, Csk did not appear to be involved in Lyn regulation and was not changed in its subcellular location. Thus, CHK may play a role both in megakaryocyte development and in platelet function. TEC Kinase The TEC tyrosine kinase, a protein of 70 kDa, is an SH2/SH3-containing kinase that is homologous to the Btk (88, 89) and Itk (90) tyrosine kinases. These kinases, unlike the Src kinases, do not contain N-terminal myristylation sites or C-terminal negative regulatory tyrosine phosphorylation sites. Also, they contain an N-terminal pleckstrin homology domain. These kinases are present primarily in hematopoietic tissues, although TEC is also present in liver tissue (91). It has recently been shown that TEC in megakaryocytic Mo7e cells is constitutively associated with c-Kit via its SH3 domain and becomes tyrosine-phosphorylated upon SCF/KL treatment of these cells (24). Similar to reports that the Lyn kinase is able to phosphorylate and activate Btk (92), there is also a report that Lyn is able to phosphorylate and activate TEC in 3T3 fibroblasts (93), indicating that TEC is downstream of Lyn in receptor signaling pathways. Although the function of TEC in megakaryocytes is not known, it is likely that it is related to proliferation as Btk deficiencies in B cells prevent these cells from proliferating and developing into antibody-secreting cells (88, 89). Related Adhesion Focal Tyrosine Kinase The related adhesion focal tyrosine kinase (RAFTK) is a novel tyrosine kinase recently identified by our laboratory (94) and by others, also referred to as PYK2 (95) and CAK-b (96). The kinase is composed of a 1009-amino-acid polypeptide and has closest homology (48% identity, 65% homology) to FAK. Like FAK, RAFTK lacks SH2/SH3 domains, myristylation sites, and transmembrane domains. RAFTK has been shown to be involved in the G-protein-linked Ca21-mediated stimulation of PC12 cells by peptide hormones, leading to the stimulation of MAP kinase (93). Members of our laboratory recently showed that RAFTK in megakaryocytic CMK cells becomes tyrosine-phosphorylated in a Ca21/protein kinase C-dependent manner in response to SCF/KL (97). RAFTK is constitutively associated with paxillin through its C-terminal

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proline-rich domain (97, 98). It was shown that tyrosine phosphorylation of paxillin was dependent on RAFTK, as expression of an RAFTK dominantnegative mutant significantly inhibited the phosphorylation of paxillin. This was an indication that signaling of SCF/KL might have part of its effect on cytoskeletal components through stimulation of RAFTK (97). We have also shown by confocal microscopy that RAFTK associates with focal adhesion-like structures in CMK cells and may participate in integrin-mediated signaling in megakaryocytes (99). Members of our laboratory have also shown the importance of RAFTK in platelet activation by thrombin (100). As in megakaryocytes, RAFTK phosphorylation in platelet activation was shown to be via a protein kinase C/Ca21-dependent pathway. As with CHK, this demonstrates that a kinase important in megakaryocyte development can also have an apparently unrelated function in the mature platelet. Focal Adhesion Kinase (FAK) The focal adhesion kinase (FAK) is a tyrosine kinase of molecular mass 125 kDa with structural properties similar to those mentioned above for RAFTK. FAK undergoes tyrosine autophosphorylation in response to the interaction of b1 and b3 integrins with extracellular matrix and plasma proteins (101–103). Cells from FAK-deficient mice show reduced motility and enhanced focal contact formation (104). In platelets, the phosphorylation of FAK is induced in response to the binding of fibrinogen to integrins aIIbb3, leading to a variety of changes within the cytoskeleton (105).

TYROSINE PHOSPHATASES IN MEGAKARYOCYTE GROWTH AND DIFFERENTIATION In comparison to tyrosine kinases, tyrosine phosphatases in megakaryocytes have been less widely studied. Classical tyrosine phosphatases have been categorized as being of two types, either receptor protein tyrosine phosphatases (RPTPs) or cytoplasmic nontransmembrane tyrosine phosphatases (non-TM PTPs) [see review by Neel and Tonks (106)]. Studies up to this point have reported four different tyrosine phosphatases in megakaryocytes that are significant in the development of these cells.

Three of these are non-TM PTPs: SHP-1, SHP-2, and fetal liver phosphatase 1 (FLP1). The fourth is an RPTP, referred to as PTP-RO, recently cloned by members of our laboratory. These phosphatases will be described in the following sections. SHP-1 and SHP-2 SHP-1 is a nontransmembrane tyrosine phosphatase of 60 kDa molecular mass. The enzyme consists of two tandem SH2 domains at the N-terminus and a catalytic domain at the C-terminus. It has been shown for a variety of activated growth factor receptor tyrosine kinases that both SHP-1 and the related SHP-2 have an affinity for tyrosine-autophosphorylated sequences (107–109). Particularly relevant to megakaryocytes is the association of SHP-1 to the activated c-Kit receptor. Yi and Ihle (110) have shown in Mo7e cells that SCF/KL stimulation leads to a time-dependent association of SHP-1 with c-Kit and that SHP-1 itself becomes phosphorylated as a result. Analogous results were found by Tauchi et al. (111). These investigators found that on SCF/KL stimulation of Mo7e cells, SHP-2 associated with c-Kit via its SH2 domain and Grb-2 was associated with SHP-1 through the tyrosine phosphorylation of SHP-1. Other investigators have recently shown that the binding of SHP-1 to c-Kit is localized to the juxtamembrane region of c-Kit at Tyr569 (112). SHP-2 was also shown to bind to c-Kit, but the site of association appears to be the neighboring Tyr567. These investigators also showed that in Ba/F3 cells overexpressing c-Kit, transfections of mutant c-Kit (Y569 3 F569) or (Y567 3 F567) produced cells that had markedly enhanced proliferation, suggesting that SHP-1 and SHP-2 might negatively regulate c-Kit function. Supporting this contention is a study from the motheaten mouse, which is deficient in SHP-1 (113). When bone marrow progenitors (lin2) are purified from me/me mice, there is a dramatic hyperproliferation in response to SCF/KL. Also, when me/me mice are crossed with Wv/Wv mice containing a mutant c-Kit, the genotypes Wv/1,me/me and Wv/Wv,me/me had decreased numbers of proliferating macrophages and granulocytes, which were hyperproliferated in 1/1,me/me mice. Erythroid cells in Wv/1,me/me were increased compared to 1/1,me/me mice where they were reduced compared to the wild-type mice. Thus, intragenic complementation by the loss of c-Kit function resulted in a lessening of the effect of the loss of SHP-1 function. This again would indicate that SHP-1 was playing an inhibitory regulatory role in c-Kit func-

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tion. Exactly which function of c-Kit is inhibited by SHP-1 is not clear. SHP-1 may dephosphorylate c-Kit autokinase sequences directly. Another possibility is that SHP-1 might dephosphorylate Src at its C-terminal negative regulatory site, resulting in higher Src kinase activity. This was supported by findings of substantially lower Src activity in the thymocytes of motheaten mice (114). Other investigators found that T cell-receptor-induced thymocytes from the motheaten mouse had higher than normal levels of Src activity and exhibited hyperproliferation three- to fivefold in response to T cell receptor signaling (115). Thus, the effect of SHP-1 may vary depending on the cell type and the particular receptors to which it is bound. Fetal Liver Phosphatase 1 Fetal liver phosphatase 1 (FLP1), a 48-kDa cytoplasmic tyrosine phosphatase, is present in a wide variety of adult hematopoietic cells (116). Progenitor K562 cells ectopically expressing functional FLP1 differentiated normally into megakaryocytes after phorbol myristic acid stimulation. However, K562 cells transfected with dominant-negative FLP1 failed to differentiate in response to TPA. It was also found that expression of a2 integrin, a late megakaryocyte marker, was suppressed in the dominant-negative expressing cells. Thus, this phosphatase appears to have a function in late-stage megakaryocyte differentiation. PTP-RO Members of our laboratory recently cloned a novel PTP cDNA from a 5-fluorouracil-treated mouse bone marrow cDNA library (117). This phosphatase, termed PTPl, has a human homolog, which we have termed PTP-RO, a 200-kDa molecular mass receptor tyrosine phosphatase that is related to the k and m receptor classes of tyrosine phosphatases. We have recently found that PTP-RO protein and mRNA are up-regulated by PMA treatment of CMS, CMK, and DAMI megakaryocyte cell lines (25). This is an indication that PTP-RO might be involved in megakaryocyte differentiation. We also found that PTP-RO is constitutively associated with c-Kit in COS-7 cells cotransfected with c-Kit and PTP-RO. PTP-RO was also associated with c-Kit in Mo7e cells. Furthermore, stimulation with SCF/KL resulted in tyrosine phosphorylation of PTP-RO. PTP-RO does not appear to dephosphorylate c-Kit,

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and it is not clear what the in vivo substrate might be. However, involvement of PTP-RO in c-Kit signaling and up-regulation of PTP-RO during megakaryocyte differentiation argue for a significant role for PTP-RO in megakaryocytopoiesis.

INTEGRATION OF RECEPTOR SIGNALING AND EFFECTS ON DOWNSTREAM PHOSPHORYLATION It has been observed in a variety of hematopoietic progenitors that stimulation of proliferation by SCF/KL is synergistic with other cytokines such as IL-3, GM-CSF, and erythropoietin (118). This cooperation has also been observed by members of our laboratory in megakaryocytic cell lines and primary cells (10). In CMK cells, the combination of SCF/KL and IL-6 was the most synergistic, while the combination of SCF/KL and GM-CSF was more or less additive. However, other investigators observed that in the Mo7e cell line, SCF/KL and GM-CSF were highly synergistic (119). It is likely that this synergism reflects the convergence of signaling pathways on a common intermediate. In the case of Mo7e cells synergized by SCF/KL and GM-CSF, it has been shown that Raf-1 is such a common signaling intermediate. In this system, both c-Kit and GM-CSF receptors interact with SOS, Grb-2, and Ras to stimulate MAP kinases (120). Other regulators such as interferon-inducible protein-10 and macrophage inflammation protein-1a have been shown to inhibit proliferation by a cyclic AMP-dependent inhibition of the Raf-1 kinase. Like GM-CSF, the cytokine thrombopoietin (TPO) induces the association of Shc/Vav with the TPO receptor, thus leading to stimulation of MAP kinase via Raf-1 (121–123). Also typical of the cytokine receptors, the TPO receptor signals the JAK/STAT pathway, leading to the activation of STAT1, STAT3, and STAT5 (121, 123). It is likely that whenever growth factor tyrosine kinase receptors and nontyrosine kinase cytokine receptors are costimulated, there is the possibility of synergistic signaling and an increase in the efficiency of signal transmission, leading to cellular growth or differentiation. Another possible role of a variety of stimuli leading to a common signaling intermediate may be that they provide a sufficient intensity or duration of MAP kinase signaling, which would be required for differentiation. It was found in stimulation of K562 cells by various mitogens that only a

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prolonged stimulation of MAP kinase resulted in differentiation (124). Thus, PMA stimulation resulted in a prolonged MAP kinase stimulation followed by differentiation. However, when this was interrupted by MEK inhibitor, differentiation failed to occur. Other stimuli of MAP kinase such as bryostatin gave short periods of MAP kinase activation without differentiation of the cells. In support of these findings, it has been found that the expression of a constitutively active MAP kinase in K562 cells also caused differentiation of the cells as had occurred when the cells were treated with PMA (125). In this context, it was shown that MAP kinase specifically induced differentiation and not the proliferation observed in other systems. Stepping back to the simultaneous effect of various growth factors and cytokines, it may be that a combination of these factors is required to give the appropriate signaling of elements such as MAP kinase, which are required for differentiation. This would also prevent the inappropriate differentiation of megakaryocytes due to a single growth factor stimulation. Most likely, complete differentiation would require an orchestration of MAP kinases, STATs, and other cellular stimuli able to bring about the development of the more mature cells that lead to the formation of platelets.

METHODS In the studies described above, a variety of cellular and molecular biology techniques were used. Many of the basic assay techniques relating to protein phosphorylation have been described in the Methods in Enzymology series (Vols. 200, 201). Thus, for techniques of protein kinase assay, protein phosphatase assay, Western blotting, immunoprecipitation, inhibitor studies, and applications of protein purification, the reader is referred to these volumes. The techniques outlined here involve some specialized procedures that have been applied to the study of hematology in general and in some cases directly to work with megakaryocyte function. Expression Systems for in Vivo Function A variety of systems have been utilized for overexpression of proteins for the determination of biological effects. We give two examples here. One involves transfection of CHK or Csk retroviral plasmids into Csk-deficient fibroblasts. The other

involves the transient cotransfection of an RAFTK expression vector along with a paxillin expression vector into COS cells. Alternatively, a dominantnegative RAFTK was used in the transfections. CHK/Csk Transfection into Csk-Deficient Cell Line To test for CHK or Csk function, Davidson et al. (82) have described a system for transfecting CHK or Csk cDNA containing retroviral vectors into a murine fibroblast that contains neither Csk nor CHK. These cells were established by Imamoto and Soriano (126) and provided by Drs. Brian Howell and Jonathan Cooper of the Fred Hutchinson Cancer Center, (Seattle, WA). cDNAs for murine p52CHK, murine p56CHK, or rat p50Csk were introduced into the multiple cloning site of the retroviral vector pBabePuro (127), thereby conferring resistance to puromycin. These constructs were then transfected into the fibroblasts by calcium phosphate precipitation (128). These transfected cells were then selected for by growth in puromycin (1.0 mg/ml) containing medium. The transformants were isolated by limiting dilution and screened via immunoblotting of cellular extracts with either CHK or Csk antibodies. For studies of cellular transformants, isolated clones were grown to confluence and harvested into a lysis buffer [20 mM Mops, pH 7.0, 150 mM NaCl, 1% Nonidet-P40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM EDTA, pH 8.0, with 10 mg each of leupeptin, aprotinin, N-tosyl-L-phenylalanine chloromethyl ketone, phenylmethylsulfonyl fluoride (PMSF), and phosphatase inhibitors, 50 mM sodium fluoride and 1 mM sodium orthovanadate]. After centrifugation, the extracts either were directly subjected to Western immunoblotting with appropriate antibodies or were immunoprecipitated with antibodies to various cellular components (i.e., cortactin, GAP, paxillin, and Shc). Lysates were also precipitated with anti-Src or anti-Fyn antibodies, after which a standard in vitro kinase assay was carried out using acid-denatured enolase as a substrate. By these methods, the authors were able to document the selective expression of CHK or Csk and to monitor the effects of these proteins on Src kinases and their substrates. This system allowed for the direct comparison of the ability of CHK vs Csk to act on Src kinases within a cellular environment. Coexpression of RAFTK and Paxillin in COS Cells Another method for expression of proteins relevant to megakaryocyte function is the use of tran-

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sient cotransfection into COS cells. Members of our laboratory (97) recently used this method to transiently cotransfect an RAFTK-expressing vector (RAFTK-pCDNA3-neo) and a paxillin vector (pRcCMV provided by R. Salgia and J. D. Griffin, Dana Farber Cancer Institute, Boston, MA) into COS cells. Using this method, we also transfected and expressed a dominant-negative RAFTK, which was constructed by carrying out a Lys475 3 Ala475 substitution. This was accomplished by the use of a site-directed mutagenesis kit (Clontech, Palo Alto, CA). Cells were transfected using the LipofectAmine reagent (Life Technologies, Gaithersburg, MD) according to the recommended conditions. After 72 h of transfection, cells were serum-starved for 4 h and treated in the absence or in the presence of PMA. Cells were extracted into a lysis buffer (50 mM Tris– HCl, pH 7.4, 1% Nonidet-P40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, leupeptin, and pepstatin). After centrifugation, lysates were immunoprecipitated with antipaxillin antibody (ICN, Costa Mesa, CA). Immunoprecipitates were washed, and SDS–PAGE transfers were blotted with anti-phosphotyrosine PY-20 antibody (ICN). Transfers were also blotted with antipaxillin antibodies. Total cell extracts were blotted with anti-RAFTK antibodies or anti-paxillin antibodies to confirm the transfection of the cells. By observing the effect of the transfection of wild-type RAFTK plus paxillin compared to dominantnegative RAFTK plus paxillin, it was possible to confirm the importance of paxillin as a substrate for RAFTK as had been suggested by studies in megakaryocytic CMK cells (97, 98). Determination of Binding Sites of SH2 Domains on Activated Receptor Tyrosine Kinases A number of techniques have been used to determine the sites of association of SH2-containing signaling molecules to phosphotyrosyl-containing sequences on receptor tyrosine kinases. These include: (i) inhibition of protein–protein interaction by synthetic phosphotyrosyl peptides; (ii) direct binding of proteins to immobilized phosphopeptides; and (iii) site-directed mutagenesis of specific tyrosines to nonphosphorylatable phenylalanines and testing the effect on binding. It should be emphasized that these methods are complementary and that the use of at least two methods is required to ensure the specificity of binding. For instance, while certain

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phosphopeptides may inhibit binding in vitro, this may not represent specific binding in vivo or the sites of phosphorylation may not be present in vivo. Thus, mutagenesis is effective in confirming the binding and ensuring that this is actually possible in vivo. In this section, we will give two examples in which the combined techniques were used for determination of the receptor binding site. These include the determination of binding of PLC-g to the FGF receptor by Peters et al. (44) and the determination of binding of CHK to c-Kit done by members of our laboratory (19). Binding of PLC-g to Phosphotyrosine-766 of FGF Receptor It had initially been shown that the FGF receptor was phosphorylated on Tyr766 and that the PLC-g– SH2 domain could bind to a proteolytic fragment of the FGF receptor that included this site. Peters et al. (44) first carried out studies of the phosphopeptide inhibition of an FGF receptor/PLC-g complex. The complex was made by immunoprecipitating the FGF receptor from a baculovirus expression system, autophosphorylating it in vitro, and then associating it with PLC-g from a BALB/c 3T3 lysate. Complexed proteins in the presence or in the absence of competing peptides were then subjected to Western blotting using an anti-PLC-g antibody. In addition to the phosphotyrosyl peptide, a nonphosphorylated and a scrambled phosphorylated peptide were used as controls. A mutant FGF receptor was constructed using an oligonucleotide containing a single T/A change that encodes the Tyr766 3 Phe766 change. Wild-type and mutant FGF receptors were then subcloned into pSV7d expression vectors and transfected into rat L6 myoblasts. Cells were selected by G418, and positive clones were identified by Western blotting with anti-FGF receptor antibodies. Serum-starved cells were then stimulated with FGF. The immunoprecipitated FGF receptor was extracted and immunoprecipitated with anti-FGF receptor antibody, and the SDS–PAGE transfers of these complexes were probed with anti-PLC-g antibodies. Other experiments showed that activation of PLC-g was also defective in these mutant FGF receptor-containing cells. Surprisingly, mitogenesis was not affected in these mutants, indicating that other pathways are involved in this function and that PLC-g may be involved more in the cytoskeletal changes that accompany differentiation.

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Binding of CHK Tyrosine Kinase to Diphosphorylated Site Tyr568/Tyr570 of Activated c-Kit Receptor Our initial work on CHK showed that it associated with c-Kit via an SH2 domain/phosphotyrosyl c-Kit interaction (83). We then formed the complex of the activated c-Kit with SH2CHK–GST fusion proteins preincubated with phosphopeptides of the various possible phosphotyrosine sequences of c-Kit (19). We found that Y721 and Y568,Y570 were two possible sites of association. When phosphopeptides including these sites were immobilized on Affi-Gel 15 beads (Bio-Rad, Richmond, CA) and used to test association with SH2CHK– GST and full-length CHK, it was found that both sites specifically associated with CHK. We then did sitedirected mutagenesis of c-Kit using a c-Kit cDNA obtained from Dr. Yosef Yarden (Weizmann Institute, Rehovot, Israel). These cDNA plasmids were mutated using appropriate oligonucleotides and the Quickchange system (Stratagene, La Jolla, CA). The mutant and wild-type cDNAs were then inserted into pcDNA3 vectors. Recombinant vectors were transfected into COS-7 cells by the LipofectAmine method (Life Technologies). After 72 h, these cells were serum-starved and stimulated with 350 ng/ml SCF/KL for 10 min. Cells were harvested and extracted into lysis buffer (20 mM Tris–HCl pH 7.4, 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10% glycerol, 1% Nonidet-P 40, 1 mM Na3VO4, 2 mM PMSF, 10 mg/ml leupeptin, and 10 mg/ml aprotinin). After centrifugation, lysis supernatants were precipitated with 5 mg of SH2CHK–GST fusion protein and glutathione–Sepharose (Pharmacia, Piscataway, NJ). Precipitates were run on SDS– PAGE, and nitrocellulose transfers were blotted with anti-c-Kit antibody. In each case, amounts of extract were normalized by cotransfection of a b-Gal expression vector and assay of b-Gal in the extracts (Promega, Madison, WI). By these techniques, we showed that only the 568Y*VY*IDPT site associated with the SH2CHK, indicating that the association of SH2CHK with the Y*MDMKPG peptide was not representative of the native protein–protein interactions in vivo. This in particular emphasizes the importance of using different methods to confirm the sites of binding of these SH2-containing proteins with activated growth factor receptors. In Vivo Phosphorylation Sites on Protein Tyrosine Kinases As mentioned in the section on sites of receptor/ SH2 domain association, it is often important to

know which receptor sites are phosphorylated in vivo and the extent to which they are phosphorylated. Most of the methodology for phosphopeptide site determination involves 32Pi in vivo labeling of proteins, followed by isolation of the protein and analysis of proteolytic fragments. Peptides have been analyzed by means of two-dimensional peptide mapping on cellulose plates, and eluted peptides have then been sequenced on automated peptide sequencers adapted for determination of 32P in individual cycles. Phosphopeptide sequences have also been determined by fast atom bombardment mass spectroscopy. These procedures have been described in great detail in the series Methods in Enzymology (Vol. 201). What will be presented here is a brief review of two examples of in vivo phosphopeptide site determination. Identification of Sites on c-Kit Phosphorylated by Protein Kinase C As mentioned previously, Blume-Jensen et al. (30) determined sites of serine/threonine phosphorylation on c-Kit by in vivo 32P-labeling of stable c-Kitexpressing porcine aortic endothelial cells. These transfected cells expressed either wild-type or mutant c-Kit, with mutations at suspected sites of phosphorylation. Mutagenesis was by a pALTER-1 vector using the Altered Sites in vitro mutagenesis system (Promega). Transfected cells were selected using geneticin. Stable cell lines expressing wildtype and mutant c-Kit were labeled with 32Pi and stimulated by SCF/KL or PMA. c-Kit was immunoprecipitated from cell lysates and subjected to 2-D tryptic peptide mapping. In the first dimension, samples were run on thin-layer electrophoresis at a pH of 1.9 and this was followed by a second dimension of thin-layer chromatography. After exposure to film, certain phosphopeptides were eluted and sequenced by Edman degradation on an Applied Biosystems Model 470A Gas Phase Sequencer. For certain peptides, phosphoamino acid analysis was conducted by a two-dimensional thin-layer method. The results of these studies showed that two of the major sites of in vivo phosphorylation were serine phosphorylations, Ser741 and Ser746. These were the same sites that were stimulated by PMA treatment of the cells and the sites were also phosphorylated by protein kinase C in vitro. Finally, site-specific mutants when mapped confirmed the identity of the sites as shown by automated sequencing. Thus, by using these techniques the authors were able to make definite conclusions about the means of down-

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regulation of c-Kit through PKC-mediated regulation. Use of Electrospray Ionization Mass Spectroscopy to Determine in Vivo Sites of Phosphorylation on Tyrosine Kinase ZAP-70 In a recent report, Watts et al. (129) have developed a novel procedure for determining the phosphate position of isolated phosphopeptides. In this technique, ZAP-70 was autophosphorylated or transphosphorylated by p56lck in vitro with [g-32P]ATP. The resulting individual phosphopeptides from a 2-D peptide map were applied to a serial microbore IMAC column followed by microbore C18 high-performance liquid chromatography. Selected peaks were then applied to electrospray mass spectroscopy on a PE Sciex APIIII mass spectrometer. After determining sites of in vitro phosphorylation based on theoretical peptide masses, the authors carried out in vivo phosphorylations by [32P]Pi labeling of Jurkat cells and then stimulated the cells with OKT3 antibody. 32P-phosphorylated ZAP-70 was isolated by immunoprecipitation and was subjected to 2-D tryptic mapping as the in vitro phosphorylated ZAP-70 had been. Spots of the in vitro and in vivo maps were correlated to identify the sites of in vivo phosphorylation. From this study, the authors showed that Tyr292 and Tyr126 are major sites of in vitro autophosphorylation of ZAP-70. In vivo, only the Tyr292 is phosphorylated in response to antigen stimulation. In vitro, p56lck phosphorylated Tyr69, Tyr178, Tyr492, and Tyr493. In vivo, Tyr492 and Tyr493 are the major corresponding phosphorylations stimulated by antigen. Thus, by this method, the authors were able to determine in vivo phosphorylations for ZAP-70. Use of Murine Transgenic Techniques for Disruption of Tyrosine Kinases Here, we will describe the use of techniques to replace functional genes with nonfunctional genes transgenically to eliminate their gene products from the cells of the developing mouse. The two examples that will be given are the Csk kinase and the CHK kinase. Csk Gene Disruption Leading to Embryonic Lethality in Mice Recently, two laboratories generated mice that were heterozygous for a mutant nonfunctional Csk

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(126, 130). These mice, when bred, produced homozygous Csk embryos that died in utero between day 9 and day 10 of gestation. In the experiments of Imamoto and Soriano (126), a defective Csk gene in a vector including a mouse PGK1 promoter and neoselectable marker was electroporated into AB1 ES cells, and clones were selected with G418. PCR was used to screen for homologous recombination and loss of the wild-type Csk gene. Csk- cells were then injected into mouse blastocysts. Chimeric mice derived from these blastocysts were then crossed with C57BL/6J mice to produce mice that were heterozygous for the nonfunctional Csk. Heterozygous mice were then bred, and offspring were analyzed for Csk expression at various stages. Embryos that failed to express Csk mRNA did not survive past embryonic day 9.5. When cells were derived from the Csk- embryos, in vitro kinase assays showed that Src kinase activity in these cells was elevated. Analysis of the C-terminal CNBr fragment of Src from these cells was carried out by in vivo 32P-labeling of the cells and by specifically immunoprecipitating pp60c-src. The immunoprecipitate was run on SDS–PAGE. Eluted pp60c-src was cleaved with CNBr (100 mg/ml in 70% formic acid), and the cleavage products were run on 27.5% polyacrylamide SDS–PAGE. This allowed the resolution of the 32P-labeled 4-kDa C-terminal tyrosine-phosphorylated Src peptide. Analysis of the Csk- cells showed greatly reduced in vivo C-terminal tyrosine phosphorylation, confirming that down-regulation of Src by C-terminal tyrosine phosphorylation did not take place in these cells. These techniques were effective in showing the significance of this Src regulation in a variety of developmental processes. Generation of CHK-Deficient Mice Another group recently developed mice that fail to express functional CHK tyrosine kinase (131), by a technique similar to that outlined above for Csk (126). To generate CHK knockout mice, a 7.5-kb portion of the 12-kb CHK genomic DNA was inserted into a PGK 1 neo cassette. TT2 ES cells were electroporated with a NotI linearized targeting vector and selected in culture by geneticin. Screening of homologous recombinants was by Southern blotting using a probe from outside the targeting vector. After recombinant cells were injected into mouse blastulas, chimeric mice were bred with C57BL/6 mice to create hybrid heterozygous mice. These mice were bred to yield the CHK- double-recombinant (CHK-deficient) mice. Unlike Csk- mice, the CHK-

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mice developed normally and were fertile. Analysis of blood and bone marrow showed no significant differences compared to wild-type mice. Analysis by FACS showed a normal distribution of bone marrow progenitors. Methyl cellulose cultures showed a normal function of colony-forming units. Observation of megakaryocytes indicated no abnormalities. In cultured bone marrow cells, there were no differences in Src, Hck, or Fgr activities compared to wild-type mice. These results, although not totally conclusive, indicate that CHK has a more narrow function compared to Csk and that Csk may be able to substitute for CHK function, where the opposite is not true, as Csk deficiency leads to lethality.

14. Miyazawa, K., Williams, D. A., Gotoh, A., Nishimata, J., Broxmeyer, H. E., and Yoyama, K. (1995) Blood 85, 641– 649. 15. Grabarek, J., Groopman, J. E., Lyles, Y. R., Jiang, S., Bennett, L., Zsebo, K., and Avraham, H. (1994) J. Biol. Chem. 269, 21718 –21724. 16. Serve, H., Hsu, Y-C., and Besmer, P. (1994) J. Biol. Chem. 269, 6026 – 6030. 17. Meisenhelder, J., Suh, P. G., Rhee, S. G., and Hunter, T. (1989) Cell 57, 1109 –1122. 18. Kypta, R. M., Goldberg, Y., Ulug, E. T., and Courtneidge, S. A. (1990) Cell 62, 481– 492. 19. Price, D. J., Rivnay, B., Fu, Y., Jiang, S., Avraham, S., and Avraham, H. (1997) J. Biol. Chem. 272, 5915–5920. 20. Kazlauskas, A., Ellis, C., Pawson, T., and Cooper, J. A. (1990) Science 247, 1578 –1581. 21. Yi, T., and Ihle, J. N. (1993) Mol. Cell. Biol. 13, 3350 –3358.

ACKNOWLEDGMENTS The authors thank Peter Park, Phuongtuong Trac, and Janet Delahanty for help in preparation of the manuscript.

REFERENCES 1. Yarden, Y., Kuang, W., Yang-Feng, T., Coussens, L., Munemitsu, S., Dull, T. J., Chen, E., Schlessinger, J., Franke, U., and Ullrich, A. (1987) EMBO J. 6, 3341–3351. 2. Qui, F., Ray, P., Brown, K., Barker, P. E., Jhanwar, S., Ruddle, F. H., and Besmer, P. (1988) EMBO J. 7, 1003–1011. 3. Sasaki, E., Okamura, H., Chikamune, T., Kanai, Y., Watanabe, S., Naito, M., and Sakurai, M., (1993) Gene 128, 257–261. 4. Kubota, T., Hikono, H., Sasaki, E., and Sakurai, M. (1994) Gene 141, 305–306. 5. Nocka, K., Tan, J., Chin, E., Chu, T. Y., Ray, P., Traktman, P., and Besmer, P. (1990) EMBO J. 9, 1805–1813. 6. Duttlinger, R., Manova, K., Berrozpe, G., Chu, T-Y., DeLeon, V., Timokhina, I., Chaganti, R. S. K., Zelenetz, A. S., Bachvarova, R. F., and Besmer, P. (1995) Proc. Natl. Acad. Sci. USA 92, 3754 –3758. 7. McCulloch, E. A., Siminovitch, L., Till, J. E., Russell, E. S., and Bernstein, S. E. (1965) Blood 26, 399 – 410. 8. Lev, S., Blechman, J. M., Givol, D., and Yarden, Y. (1994) Crit. Rev. Oncogenesis 5, 141–168. 9. Wershil, B. K., Tsai, M., Geissler, E. N., Zsebo, K. M., and Galli, S. J. (1992) J. Exp. Med. 175, 245–255. 10. Avraham, H., Vannier, E., Cowley, S., Jiang, S., Chi, S., Dinarello, C. A., Zsebo, K. A., and Groopman, J. E. (1992) Blood 79, 365–371. 11. Hassan, H. T., and Freund, M. (1995) Leukemia Res. 19, 589 –594. 12. Imai, T., and Nakahata, T. (1994) Int. J. Hematol. 59, 91–98. 13. Avraham, H., Scadden, D. T., Chi, S., Broudy, V. C., Zsebo, K. M., and Groopman, J. E. (1992) Blood 7, 1679 –1684.

22. Lorenz, U., Bergemann, A. D., Steinberg, H. N., Flanagan, J. G., Li, X., Galli, S. J., and Neel, B. G. (1996) J. Exp. Med. 184, 1111–1126. 23. Paulson, R. F., Vesely, S., Siminovitch, K. A., and Bernstein, A. (1996) Nat. Genet. 13, 309 –315. 24. Tang, B., Mano, H., Yi, T., and Ihle, J. N. (1994) Mol. Cell. Biol. 14, 8432– 8437. 25. Taniguchi, Y., Avraham, S., London, R., Jiang, S., and Avraham, H., submitted for publication. 26. Kinashi, T., Escobedo, J. A., Williams, L. T., Takatsu, K., and Springer, T. A. (1995) Blood 86, 2086 –2090. 27. Courtneidge, S. A., Dhand, R., Pilat, D., Twamley, G. M., Waterfield, M. D., and Roussel, M. F. (1993) EMBO J. 12, 943–950. 28. Blume-Jensen, P. Siegbahn, A., Stabel, S., Heldin, C.-H., and Ronnstrand, L. (1993) EMBO J. 12, 4199 – 4209. 29. Blume-Jensen, P., Ronnstrand, L., Gout, I., Waterfield, M. D., and Heldin, C.-H. (1994) J. Biol. Chem. 269, 21793– 21802. 30. Blume-Jensen, P., Wernstedt, C., Heldin, C.-H., and Ronnstrand, L. (1995) J. Biol. Chem. 270, 14192–14200. 31. Lee, P. L., Johnson, D. E., Coussens, L. S., Fried, V. A., and Williams, L. T. (1989) Science 245, 57– 60. 32. Dionne, C. A., Crumley, G., Bellot, F., Kaplow, J. M., Searfoss, G., Ruta, M., Burgess, W. H., Jaye, M., and Schlessinger, J. (1990) EMBO J. 9, 2685–2692. 33. Keegan, K., Johnson, D. E., Williams, L. T., and Hayman, M. J. (1991) Proc. Natl. Acad. Sci. USA 88, 1095–1099. 34. Partanen, J., Makela, T. P., Eerola, E., et al. (1991) EMBO J. 10, 1347–1354. 35. Rifkin, D. B., and Moscatelli, D. (1989) J. Cell Biol. 109, 1– 6. 36. Gabbianelli, M., Sargiacomo, M., Pelosi, E., Testa, U., Isacchi, G., and Peschle, C. (1990) Science 249, 1561–1564. 37. Gallicchio, V. S., Hughes, N. K., Hulette, B. C., DellaPuca, R., and Noblitt, L. (1991) Int. J. Cell Cloning 9, 220 –232. 38. Thomas, K. A., Rios-Candelore, M., Gimenez-Gallego, G., DiSalvo, J., Bennett, C., Rodkey, J., and Fitzpatrick, S. (1985) Proc. Natl. Acad. Sci. USA 82, 6409 – 6413. 39. Oliver, L. J., Rifkin, D. B., Gabrilove, J., Hannocks, M. J., and Wilson, E. L. (1990) Growth Factors 3, 231–236.

MEGAKARYOCYTOPOIESIS, PLATELET PRODUCTION, AND TYROSINE PHOSPHORYLATION 40. Wilson, E. L., Rifkin, D. B., Kelly, F., Hannocks, M. J., and Gabrilove, J. (1991) Blood 77, 954 –960. 41. Brunner, G., Nguyen, H., Gabrilove, J., Rifkin, D. B., and Wilson, E. L. (1993) Blood 81, 631– 638. 42. Bikfalvi, A., Han, Z. C., and Fuhrmann, G. (1992) Blood 80, 1905–1913. 43. Avraham, H., Banu, N., Scadden, D. T., Abraham, J., and Groopman, J. E. (1994) Blood 83, 2126 –2132. 44. Peters, K. G., Marie, J., Wilson, E., Ives, H. E., Escobedo, J., Del Rosario, M., Mirda, D., and Williams, L. T. (1992) Nature 358, 678 – 681.

263

65. Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. USA 90, 7696 –7700. 66. Roche, S., Koegle, M., Barone, M. V., Roussel, M. F., and Courtneidge, M. F. (1995) Mol. Cell. Biol. 5, 1102–1109. 67. Alonso, G., Koegle, M., Mazurenko, N., and Courtneidge, S. A. (1995) J. Biol. Chem. 270, 9840 –9848. 68. Landgren, E., Blume-Jensen, P., Courtneidge, S. A., and Claesson-Welsh, L. (1995) Oncogene 10, 2027–2035. 69. Barone, M. V., and Courtneidge, S. A. (1995) Nature 378, 509 –512.

45. Mohammadi, M. Dionne, C. A., Li, W., Li, N., Spivak, T., Honegger, A. M., Jaye, M., and Schlessinger, J. (1992) Nature 358, 681– 684.

70. Li, Y., Karanes, C., Sensenbrenner, L., and Chen, B. (1995) J. Immunol. 154, 2165–2174.

46. Huang, J., Mohammadi, M., Rodrigues, G. A., and Schlessinger, J. (1995) J. Biol. Chem. 270, 5065–5072.

72. Bennett, B. D., Cowley, S., Jiang, S., London, R., Deng, B., Grabarek, J., Groopman, J. E., Goeddel, D. V., and Avraham, H. (1994) J. Biol. Chem. 269, 1068 –1074.

47. Tang, T. L., Freeman, R. M., O’Reilly, A. M., Neel, B. G., and Sokol, S. Y. (1995) Cell 80, 473– 483. 48. Saxton, T. M., Henkemeyer, M., Gasca, S., Shen, R., Rossi, D. J., Shalaby, F., Feng, G. S., and Pawson, T. (1997) EMBO J. 16, 2352–2364. 49. Ronsin, C., Muscatelli, F., Mattei, M. G., and Breathnach, R. (1993) Oncogene 8, 1195–1198. 50. Huff, J. L., Jelinek, M. A., Borgman, C. A., Lansing, T. J., and Parsons, J. T. (1993) Proc. Natl. Acad. Sci. USA 90, 6140 – 6144. 51. Skeel, A., and Leonard, E. J. (1994) J. Immunol. 152, 4618 – 4623. 52. Leonard, E. J., and Skeele, A. (1976) Exp. Cell Res. 114, 434 – 438. 53. Leonard, E. J., and Skeele, A., (1978) Exp. Cell Res. 114, 117–126. 54. Banu, N., Price, D. J., London, R., Deng, B., Mark, M., Godowski, P., and Avraham, H. (1996) J. Immunol. 156, 2933–2940. 55. Wang, M. H., Montero-Julian, F. A., Dauny, I., and Leonard, E. J. (1996) Oncogene 13, 2167–2175. 56. Iwana, A. M., Yamaguchi, N., and Suda, T. (1996) EMBO J. 15, 5866 –5875. 57. Broxmeyer, H. E., Cooper, S., Li, Z. H., Lu, L., Sarris, A., Wang, M. H., Chang, M. S., Donner, D. B., and Leonard, E. J. (1996) Ann. Hematol. 73, 1–9. 58. McArthur, G. A., Rohrschneider, L. R., and Johnson, G. R. (1994) Blood 83, 972–981. 59. Byrne, P. V., Guilbert, L. J., and Stanley, E. R. (1981) J. Cell. Biol. 91, 848 – 853.

71. Ihle, J. N. (1995) Nature 377, 591–594.

73. Avraham, S., Jiang, S., Ota, S., Fu, Y., Deng, B., Dowler, L. L., White, R. A., and Avraham, H. (1995) J. Biol. Chem. 270, 1833–1842. 74. Sakano, S., Iwama, A., Inazawa, J., Ariyama, T., Ohno, M., and Suda, T. (1994) Oncogene 9, 1155–1161. 75. Chow, L. M. L., Jarvis, C., Hu, Q., Nye, S. H., Gervais, F. G., Veillette, A., and Matis, L. A. (1994) Proc. Natl. Acad. Sci. USA 91, 4975– 4979. 76. Chow, L. M. L., Davidson, D., Fournel, M., Gosselin, P., Lemieux, S., Lyu, M. S., Kozak, C. A., Matis, L. A., and Veillette, A. (1994) Oncogene 9, 3437–3448. 77. Klages, S., Adam, D., Class, K., Fargnoli, J., Bolen, J. B., and Penhallow, R. C. (1994) Proc. Natl. Acad. Sci. USA 91, 2597–2601. 78. Kuo, S. S., Moran, P., Gripp, J., Armanini, M., Phillips, H. S., Goddard, A., and Caras, I. W. (1994) J. Neurosci. Res. 38, 705–715. 79. McVicar, D. W., Lal, B. K., Lloyd, A., Kawamura, M., Chen, Y.-Q., Zhang, X., Staples, J. E., Ortaldo, J. R., and O’Shea, J. J. (1994) Oncogene 9, 2037–2044. 80. Grgurevich, S., Linnekin, D., Musso, T., Zhang, X., Modi, W., Varesio, L., Ruscetti, F. W., Ortaldo, J. R., and McVicar, D. W. (1997) Growth Factors 14, 103–115. 81. Zrihan-Licht, S., Lim, J., Keydar, I., Sliwkowski, M. X., Groopman, J. E., and Avraham, H. (1997) J. Biol. Chem. 272, 1856 –1863. 82. Davidson, D., Chow, L. M. L., and Veillette, A. (1997) J. Biol. Chem. 272, 1355–1362.

60. Motoyoshi, K. (1994) Oncology 51, 198 –204.

83. Jhun, B. H., Rivnay, B., Price, D. J., and Avraham, H. (1995) J. Biol. Chem. 270, 9661–9666.

61. Sherr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F., Look, A. T., and Stanley, E. R. (1985) Cell 41, 665– 676.

84. Zrihan-Licht, S., Deng, B., Yarden, Y., McShan, G., Keydar, I., and Avraham, H. (1998) J. Biol. Chem. 273, 4065– 4072.

62. Sherr, C. J., and Stanley, E. R. (1990) in Peptide Growth Factors and Their Receptors. (Sporn, M. B., and Roberts, A. B., Eds.), pp. 667– 698, Springer-Verlag, Heidelberg.

85. Yamashita, H., and Avraham, H., submitted for publication.

63. Hanks, S. J., Quinn, A. M., and Hunter, T., (1988) Science 241, 42–52.

87. Hirao, A., Hamaguchi, I., Suda, T., and Yamaguchi, N. (1997) EMBO J. 16, 2342–2351.

64. Kovacs, C. J., Powell, D. S., Evans, M. J., Thomas-Patterson, D., and Johnke, R. M. (1996) J. Interferon Cytokine Res. 16, 187–194.

88. Tsukada, S. Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R. C., Klisak, I., Kubagawa, H., Mohandas, T., and Witte, O. N. (1993) Cell 72, 279 –290.

86. Price, D. J., Raychaudhuri, A., and Avraham, H. (1997) Blood 90, 725.

264

AVRAHAM AND PRICE

89. Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., Smith, C. I. E., and Bentley, D. R. (1993) Nature 361, 226 –233. 90. Silicano, J. D., Morrow, T. A., and Desiderio, S. V. (1992) Proc. Natl. Acad. Sci. USA 89, 11194 –11198. 91. Mano, H., Ishikawa, F., Nishida, J., Hirai, H., and Takaku, F. (1993) Oncogene 8, 417– 424. 92. Rawlings, D. J., Scharenberg, A. M., Park, H., Wahl, M. I., Lin, S., Kato, R. M., Fluckiger, A.-C., Witte, O. N., and Kinet, J.-P. (1996) Science 271, 822– 825. 93. Mano, H., Yamashita, Y., Miyazata, A., Miura, Y., and Ozawa, K. (1996) FASEB J. 10, 637– 642. 94. Avraham, S., London, R., Fu, Y., Ota, S., Hiregowdara, D., Li, J., Jiang, S., Pasztor, L. M., White, R. A., Groopman, J. E., and Avraham, H. (1995) J. Biol. Chem. 270, 27742– 27751. 95. Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737–744. 96. Sasaki, H., Nagura, K., Ishino, M., Tobioka, H., Kotani, K., and Sasaki, T. (1995) J. Biol. Chem. 270, 21206 –21219. 97. Hiregowdara, D., Avraham, H., Fu, Y., London, R., and Avraham, S. (1997) J. Biol. Chem. 272, 10804 –10810. 98. Salgia, R., Avraham, S., Pisick, E., Li, J. L., Raja, S., Greenfield, E. A., Sattler, M., Avraham, H., and Griffin, J. D. (1996) J. Biol. Chem. 271, 31222–31226. 99. Li, J., Avraham, H., Rogers, R., Raja, S., and Avraham, S. (1996) Blood 88, 417– 428. 100. Raja, S., Avraham, S., and Avraham, H. (1997) J. Biol. Chem. 272, 10941–10947. 101. Juliano, R. (1996) BioEssays 18, 911–917. 102. Hanks, S. K., Calalb, M. B., Harper, M. C., and Patel, S. K. (1992) Proc. Natl. Acad. Sci. USA 89, 8487– 8491. 103. Zachary, I., and Rozengurt, E. (1992) Cell 71, 891– 894. 104. Ilic, D., Furuta, Y. Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., Yamamoto, T., and Izawa, S. (1995) Nature 377, 539 –544. 105. Shattil, S. J., Haimovich, B., Cunningham, M., Lipfert, L., Parsons, J. T., Ginsberg, M. H., and Brugge, J. S. (1994) J. Biol. Chem. 269, 14738 –14745. 106. Neel, B. G., and Tonks, N. K. (1997) Curr. Opin. Cell Biol. 9, 193–204. 107. Paulson, R. F., Vesely, S., Siminovitch, K. A., and Bernstein, A. (1996) Nat. Genet. 13, 309 –315. 108. Perkins, L. A., Larsen, I., and Perrimon, N. (1992) Cell 70, 225–236. 109. Bennett, A. M., Hausdorff, S. F., O’Reilly, A. M., Freeman, R. M. Jr., and Neel, B. G. (1995) Mol. Cell. Biol. 16, 1189 – 1202. 110. Yi, T., and Ihle, J. N. (1993) Mol. Cell. Biol. 13, 3350 –3358.

111. Tauchi, T., Feng, G. S., Marshall, M. S., Shen, R., Mantel, C., Pawson, T., and Broxmeyer, H. E. (1994) J. Biol. Chem. 269, 25206 –25211. 112. Kozlowski, M., Larose, L., Lee, F., Mingh Le, D., Rottapel, R., and Siminovitch, K. A. (1998) Mol. Cell. Biol. 18, 2089 –2099. 113. Lorenz, U., Bergemann, A. D., Steinberg, H. N., Flanagan, J. G., Li, X., Galli, S. J., and Neel, B. G. (1996) J. Exp. Med. 184, 1111–1126. 114. Somani, A.-K., Bignon, J. S., Mills, G. B., Siminovitch, K. A., and Branch, D. R. (1997) J. Biol. Chem. 272, 21113–21119. 115. Lorenz, U., Ravichandran, K. S., Burakoff, S. J., and Neel, B. G. (1996) Proc. Natl. Acad. Sci. USA 93, 9624 –9629. 116. Dosil, M., Leibman, N., and Lemischka, I. R. (1996) Blood 88, 4510 – 4525. 117. Avraham, S., London, R., Tulloch, G. A., Ellis, M., Fu, Y., Jiang., S., White, R. A., Painter, C., Steinberger, A. A., and Avraham, H. (1997) Gene 204, 5–16. 118. Broxmeyer, H. E., Hangoc, G., Cooper, S., Ribeiro, R. C., Graves, V., Yoder, M., Wagner, J., Vadhan-Raj, S., Benninger, L., Rubinstein, P., and Broun, E. R. (1992) Proc. Natl. Acad. Sci. USA 89, 4109 – 4113. 119. Mantel, C., Luo, Z., Canfield, J., Braun, S., Deng, C., and Broxmeyer, H. E. (1996) Blood 88, 3710 –3719. 120. Aronica, S. M., Mantel, C., Gonin, R., Marshall, M. S., Sarris, A., Cooper, S., Hague, N., Zhang, X-f., and Broxmeyer, H. E. (1995) J. Biol. Chem. 270, 21998 –22007. 121. Nagata, Y., and Todokoro, K. (1995) FEBS Lett. 377, 497– 501. 122. Yamada, M., Komatsu, N., Okada, K., Kato, T., Miyazaki, H., and Miura, Y. (1995) Biochem. Biophys. Res. Commun. 217, 230 –237. 123. Nagata, Y., Nagahisa, H., Nagasawa, Y., and Todokoro, K. (1997) Leukemia 11(Suppl. 3), 435– 438. 124. Racke, F. K., Lewandowska, K., Goueli, S., and Goldfarb, A. N. (1997) J. Biol. Chem. 272, 23366 –23370. 125. Whelan, A. M., Galasinski, S. C., Shapiro, P. S., Nahreini, T. S., and Ahn, N. G. (1997) Mol. Cell. Biol. 17, 1947–1958. 126. Imamoto, A., and Soriano, P. (1993) Cell 73, 1117–1124. 127. Morgenstern, J. P., and Land, H. (1990) Nucleic Acids Res. 18, 3587–3596. 128. Cartier, M., Chang, M. W. M., and Stanners, C. P. (1987) Mol. Cell. Biol. 7, 1623–1628. 129. Watts, J. D., Affolter, M., Krebs, D., Wange, R. L., Samelson, L. E., and Aebersold, R. (1994) J. Biol. Chem. 269, 29520 – 29529. 130. Nada, S., Yagi, T., Takeda, H., Tokunaga, T., Nakagawa, H., Ikawa, Y., Okada, M., and Aizawa, S. (1993) Cell 73, 1125– 1135. 131. Hamaguchi, I., Yamaguchi, N., Suda, J., Iwama, A., Hirao, A., Hashiyama, M., Aizawa, S.-I., and Suda, T. (1996) Biochem. Biophys. Res. Commun. 224, 172–179.